Tyrphostin analogs are GPR35 agonists

FEBS Letters 585 (2011) 1957–1962

journal homepage: www.FEBSLetters.org

Tyrphostin analogs are GPR35 agonists Huayun Deng 1, Haibei Hu 1, Ye Fang ⇑ Biochemical Technologies, Science and Technology Division, Corning Inc., Corning, NY 14831, United States

a r t i c l e

i n f o

Article history: Received 14 April 2011 Revised 7 May 2011 Accepted 11 May 2011 Available online 17 May 2011 Edited by Christian Griesinger

a b s t r a c t GPR35 is an orphan G protein-coupled receptor that is not well-characterized. Here we employ dynamic mass redistribution (DMR) assays to discover new GPR35 agonists. DMR assays identified tyrphostin analogs as GPR35 agonists, which were confirmed with receptor internalization, Tango b-arrestin translocation, and extracellular-signal-regulated kinase phosphorylation assays. These agonists provide pharmacological tools to study the biology and function of GPR35. Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Dynamic mass redistribution GPR35 G protein-coupled receptor Optical biosensor Tyrphostin

1. Introduction GPR35 was first identified to be a class A orphan G proteincoupled receptor (GPCR) that contains 309 amino acids [1]. GPR35b, a splicing variant that contains an N-terminal extension of 31 amino acids, was later discovered in gastric cancer cells in 2004, and shown to be capable of transforming NIH-3T3 cells [2]. GPR35 has been found to be expressed in various tissues including stomach [2], gastrointestinal tissues [3], and mast cells, basophils and eosinophils [4]. Upregulation of GPR35 has also been identified in human mast cells upon challenge with IgE antibodies [4], in human macrophages after exposure to benzo(a)pyrene [5], in failing heart cells [6], and in gastric cancer cells [2]. To date, both kynurenic acid and 2-acyl lysophosphatidic acids have been postulated to be natural agonists for GPR35 [7,8]. Both ligands elicited cellular responses via GPR35 in engineered cells expressing GPR35. However, it remains controversial whether both agonists are true endogenous agonists, and it is largely unknown about the biological functions of GPR35 [9]. Thus, identification of new classes of ligands would be beneficial to elucidate the biology and pharmacology of GPR35. Here, we applied a pathway-unbiased, but pathway-sensitive technology, dynamic mass Abbreviations: DMR, dynamic mass redistribution; GPCR, G protein-coupled receptor; RWG, resonant waveguide grating; ERK, extracellular-signal-regulated kinase; CMOT, catechol-O-methyl transferase ⇑ Corresponding author. Fax: +1 607 974 5957. E-mail address: [email protected] (Y. Fang). 1 Equal contributions.

redistribution (DMR) assay [10], to discover new GPR35 ligands. The DMR assay is enabled by label-free resonant waveguide grating (RWG) biosensor [11], and has attracted much interest in the molecular delineation of receptor biology and ligand pharmacology [12–14]. DMR agonism screening in HT-29, a human colon cancer cell line that endogenously expresses GPR35, led to identification of tyrphostin analogs including entacapone as GPR35 agonists. 2. Materials and methods 2.1. Materials and cells Tyrphostins and entacapone were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Zaprinast and BioMol 80 kinase inhibitor library was purchased from Enzo Life Sciences (Plymouth Meeting, PA). The InhibitorSelect™ 96-well protein kinase inhibitor library I (Cat. No. 539744) and II (Cat. No. 539745) were purchased from EMD Chemicals (Gibbstown, NJ, USA). All inhibitors were stocked in dimethyl sulfoxide (DMSO) at 10 mM, and were diluted directly in the assay buffer (1 Hanks’ balanced salt buffer, 20 mM Hepes, pH 7.1; HBSS) to the indicated concentrations. EpicÒ 384-well biosensor microplates were obtained from Corning Inc. (Corning, NY, USA). Mouse monoclonal anti-phosphorylated extracellular-signal-regulated kinase 1/2 (anti-pERK1/2) (Cat. #M9682) and mouse monoclonal anti-ERK1/2 (#M7431) were obtained from Sigma. Goat anti-GPR35 antibody (#T14, cytoplasmic) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.05.026

1958

H. Deng et al. / FEBS Letters 585 (2011) 1957–1962

Alexa FluorÒ 594 donkey anti-goat IgG (H + L) was purchased from Invitrogen (Carlsbad, CA, USA). Human colorectal adenocarcinoma HT-29 was obtained from American Type Cell Culture (Manassas, VA). The cells were cultured in McCoy’s 5a Medium Modified supplemented with 10% fetal bovine serum, 4.5 g/l glucose, 2 mM glutamine, and antibiotics at 37 °C under air/5% CO2. Tango™ GPR35-bla U2OS cells were obtained from Invitrogen. Tango™ GPR35-bla U2OS cells contain the human GPR35 linked to a TEV protease site and a Gal4-VP16 transcription factor stably integrated into the Tango™ GPR35-bla U2OS parental cell line. This parental cell line stably expresses a b-arrestin/TEV protease fusion protein and the b-lactamase reporter gene under the control of a UAS response element. The cells were cultured according to the protocols recommended by the supplier. Briefly, the cells were passed using McCoy’s 5A medium (Invitrogen 16600-082) supplemented with 10% dialyzed fetal bovine serum, 0.1 lM NEAA, 25 lM Hepes (pH 7.3), 1 mM sodium pyruvate, 100 U/ml penicillin, 100 lg/ml streptomycin, 200 lg/ml zeocin, 50 lg/ml hygromycin, and 100 lg/ml geneticin in a humidified 37 °C/5% CO2 incubator.

2.3. Receptor internalization assays HT29 cells were plated on an 8-well chamber slide with a seeding density of 10 000 cells/well and incubated at 37 °C for 24 h. Next day, cells were stimulated with a compound or equal amount of DMSO at 37 °C for 1 h. Afterwards, cells were fixed with 4% formaldehyde in 1 PBS for 15 min, followed by blocking and permeabilization in a buffer containing 4% goat serum, 0.1% bovine serum albumin (BSA), 0.1% Triton X100 in 1 PBS for 2 h. After 5 min wash with PBS, fixed cells were incubated with the anti-GPR35 (1:500) in 3% BSA/PBS buffer for 24 h, followed by incubation with secondary antibody Alexa FluorÒ 594 donkey anti-goat IgG (H + L) (1:500) in 3% BSA/PBS for 1 h at room temperature. Cells were finally washed once with PBS and sealed with 1.5 mm thick glass cover-slip. Dried slides were stored at 4 °C until imaging. Confocal imaging was performed with Zeiss confocal microscope Axiovert 40. Confocal images were analyzed using MacBiophotonics Image J software (http:// www.macbiophotonics.ca/downloads.htm). 2.4. ERK MAPK assays

2.2. DMR assays using EpicÒ system All DMR assays were performed using EpicÒ system (Corning Inc.). EpicÒ is a wavelength interrogation reader system tailored for RWG biosensor in microtiter plates. This system consists of a temperature-control unit (26 °C), an optical detection unit, and an on-board liquid handling unit with robotics. The detection unit is centered on integrated fiber optics, and enables kinetic measures of cellular responses with a time interval of 15 s. Cells were directly seeded in EpicÒ plates and cultured overnight to form confluent monolayer in the cell culture medium. After being washed twice, the cells were maintained with HBSS and further incubated inside the system for 1 h. For agonism screen, a 2-min baseline was then established. Immediately after the compound addition using the onboard liquid handler, the cellular responses were recorded. For desensitization assays, cells were initially treated with compounds for 1 h, followed by stimulation with zaprinast at a fixed dose. The cellular responses were recorded throughout the assays. All EC50 or IC50 described in the main text were calculated based on the amplitudes of DMR signals at 8 min post-agonist stimulation. Since all GPR35 agonists led to a sustained positive-DMR (PDMR) signal, the amplitudes at 50 min post-stimulation were also used to determine kinetics dependent potency and efficacy of all ligands (see Table 1).

The p44/42 MAP kinases were examined using Western blotting. Whole cell lysates were collected after the cells were treated with a compound or DMSO for 1 h. Equivalent gel loading was confirmed by probing with anti-actin body. The total ERK1/2 and phosphorylated ERK1/2 were blotted using respective antibodies. 2.5. Tango™ b-arrestin assays The Tango™ GPR35 b-arrestin assay takes advantage of GPR35 agonist-induced recruitment of protease tagged beta-arrestin molecules to GPR35 that has been modified at the C-terminus to include a transcription factor linked by a protease cleavage site. As a result of arrestin recruitment, the protease cleaves the transcription factor from the receptor, which then translocates to the nucleus and activates the expression of beta-lactamase. The assay protocol recommended by the supplier was used. Briefly, 10 000 cells/well were seeded in 384-well, black-wall, clear bottom assay plates with low fluorescence background (Corning), and cultured in DMEM (Invitrogen, 10569-010) supplemented with 10% dialyzed fetal bovine serum, 0.1 lM NEAA, 25 lM Hepes (pH 7.3), 100 U/ml penicillin, and 100 lg/ml streptomycin. After overnight culture, the cells were stimulated with ligands for 5 h in a humidified 37 °C/5% CO2, and then loaded

Table 1 Compound name, and its efficacy relative to the maximal response induced by zaprinast, EC50 at 8 min or 50 min post-stimulation, apparent IC50 to desensitize the cells, all of which were obtained using DMR assays. EC50 obtained using Tango assays were also included. Compound

Zaprinast Tyrphostin-1 Tyrphostin-9 Tyrphostin-23 Tyrphostin-25 Tyrphostin-46 Tyrphostin-47 Tyrphostin-51 AG-126 AG-490 AG-494 AG-825 AG-1024 AG-1288 Entacapone

EC50 (lM)

EC50 (lM)

P-DMR (8 min)

P-DMR (50 min)

P-DMR (8 min)

P-DMR (50 min)

100 Inactive 11 ± 3 94 ± 4 107 ± 9 74 ± 6 80 ± 5 98 ± 4 54 ± 13 Inactive 28 ± 1 55 ± 8 40 ± 10 78 ± 2 97 ± 3

100 Inactive 63 ± 1 109 ± 5 102 ± 8 83 ± 5 101 ± 6 99 ± 9 97 ± 15 Inactive 95 ± 3 65 ± 13 117 ± 11 80 ± 7 140 ± 5

0.16 ± 0.02 Inactive n.a. 3.0 ± 0.3 0.94 ± 0.07 4.6 ± 0.2 3.9 ± 0.2 0.19 ± 0.02 11.9 ± 0.4 Inactive n.a. 15.2 ± 3.1 n.a. 0.44 ± 0.04 6.3 ± 0.7

0.05 ± 0.01 Inactive 1.0 ± 0.2 2.0 ± 0.2 0.70 ± 0.05 1.9 ± 0.2 2.8 ± 0.4 0.11 ± 0.03 10.2 ± 0.9 Inactive 12.5 ± 2.1 9.5 ± 2.3 2.4 ± 1.2 0.21 ± 0.04 31.7 ± 4.1

% zaprinast at EC100

IC50 (lM)

EC50 (lM) (Tango)

0.32 ± 0.04 Inactive Inactive 4.0 ± 0.5 0.94 ± 0.07 5.4 ± 0.6 2.7 ± 0.3 0.21 ± 0.05 27.1 ± 3.0 Inactive 35 ± 7 16.1 ± 4.1 >32 0.70 ± 0.06 6.6 ± 0.9

6.2 ± 0.9 Inactive Inactive >60 5.3 ± 1.1 >60 19.2 ± 3.1 7.7 ± 0.9 Inactive Inactive Inactive Inactive Inactive 25.3 ± 4.7 Inactive

1959

H. Deng et al. / FEBS Letters 585 (2011) 1957–1962

with the cell permeable LiveBLAzer™ FRET B/G substrate. After the 2 h incubation the coumarin:fluorescein ratio was measured using Tecan Safire II microplate reader (Männedorf, Switzerland). The FRET B/G substrate contains two fluorophores, coumarin and fluorescein, in which excitation of the coumarin results in fluorescence resonance energy transfer to the fluorescein moiety and emission of green fluorescent light. In the absence of beta-lactamase expression (i.e., no GPR35 activation), cells generate green fluorescence. In the presence of beta-lactamase expression upon receptor activation, the substrate is cleaved and the cells generate blue fluorescence. The coumarin:fluorescein ratio was used as a normalized reporter response. 3. Results 3.1. Characteristics of known GPR35 agonists in HT29 To determine the presence of functional GPR35 in HT29, we first examined the DMR responses induced by three known GPR35 agonists including zaprinast [15], pamoic acid [16], and kynurenic acid [7]. Results showed that all three ligands gave rise to a dose-dependent and saturable response (Fig. 1a); and the EC50 values were found to be 2.1 ± 0.2 nM (n = 4), 0.16 ± 0.02 lM (n = 4), and 152 ± 17 lM (n = 4) for pamoic acid, zaprinast, and kynurenic acid, respectively (Fig. 1b). All three agonists also dose-dependently desensitized the cells to the repeated stimulation with 1 lM zaprinast, leading to an apparent IC50 of

3.9 ± 0.2 nM (n = 4), 1.22 ± 0.10 lM (n = 4), and 234 ± 25 lM (n = 4) for pamoic acid, zaprinast, and kynurenic acid, respectively (Fig. 1c). Immunostaining with anti-GPR35 showed that GPR35 was presented and located primarily at the cell plasma membrane in unstimulated HT29 cells (Fig. 1d), and got partially internalized by 10 lM pamoic acid (Fig. 1e). Together, these results suggest that GPR35 in HT29 is functional, and all three ligands are indeed agonists for GPR35. 3.2. DMR agonism screen Next, we screened three kinase libraries that include a total of 240 known kinase inhibitors, using DMR agonism assay. Each inhibitor was assayed with four replicates. Zaprinast was included as a positive control. Hits were selected based on their maximal responses within 15 min post-stimulation that are greater than 30% of the zaprinast response. Twenty-five hits were identified (Fig. 2). Follow-up structure activity analysis showed that 11 out of the 25 hits are tyrphostins, including AG-112, AG-1024, tyrphostin-51, tyrphostin-25, tyrphostin-47, AG-370, AG-1288, tyrphostin-23, AG-126, tyrphostin-46, and AG-825 (Fig. 3). Further, AG-494, AG-879, tyrphostin-9, and quercetin also led to noticeable DMR. The other five tyrphostins including AG-490, AG-1478, AG-1295, AG-1296, and tyrphostin-1 in the libraries did not trigger any noticeable DMR. Quercetin was recently identified to be a GPR35 partial agonist [17]. These results suggest that tyrphostins are possible GPR35 agonists.

b

a

400

Response (pm)

200 500µM 250µM 1000µM 125µM

150 100

62.5µM 31µM 16µM

50 0

Response (pm)

250

10

20

30

40

Response (pm)

400 300

100

-100 -10

50

-9

-8

-7

-6

-5

-4

-3

Compound, log M

Time (min)

c

200

0

-50 0

300

Zaprinast kynurenic acid Pamoic acid

d

e

Zaprinast kynurenic acid Pamoic acid

200 100 0 -100 -10

-9

-8

-7

-6

-5

-4

-3

Compound, log M Fig. 1. Characterization of three known GPR35 agonists including zaprinast, pamoic acid and kynurenic acid. (a) The dose DMR responses of kynurenic acid in HT29. (b) The DMR amplitudes of three agonists as a function of their doses. (c) The DMR amplitudes of 1 lM zaprinast as a function of doses of three agonists. Here the cells were prestimulated with distinct agonists at different doses for 1 h. The amplitudes at 8 min post-stimulation were used to calculate their potency for (b) and (c). (d) The confocal image of unstimulated HT29 cells. (e) The confocal image of HT29 cells 1 h after stimulated with 10 lM pamoic acid. The images were obtained after the cells were permeabilized and stained with anti-GPR35, followed staining with the fluorescent secondary antibody.

1960

H. Deng et al. / FEBS Letters 585 (2011) 1957–1962

(CMOT) inhibitor for the treatment of Parkinson’s disease [18]. Thus, we characterized entacapone using DMR assays. Results showed that entacapone led to a dose-dependent DMR with an EC50 of 6.3 ± 0.7 lM (n = 4), whose DMR characteristics is similar to other GPR35 agonists including zaprinast and tyrphostin-51 (Fig. 4b). Similar to tyrphostin-51, entacapone also dose-dependently desensitized the cells responding to the repeated stimulation with 1 lM zaprinast, leading to an apparent IC50 of 31.7 ± 4.1 lM (n = 4) (Fig. 4c). These results suggest that entacapone may also be a GPR35 agonist.

400

Response (pm)

Zaprinast 200

DMSO 0

-200 -40

0

40

80

120

160

200

3.4. GPR35 internalization, ERK phosphorylation and arrestin translocation induced by tyrphostins

240

Compound Fig. 2. The DMR responses as a function of compounds. Each compound was assayed at four replicates. The DMR amplitudes of all compounds at 8 min postsimulation were plotted.

3.3. Dose-dependent DMR signals of tyrphostins The three kinase libraries consist of 19 tyrphostin analogs, 14 of which led to detectable DMR when being screened at 10 lM. Thus, we were focused on characterization of tyrphostins. We first examined 13 tyrphostins for their dose-dependent responses in HT29 cells using DMR assays. Results showed that except for tyrphostin1 and AG-490, all tyrphostins tested gave rise to dose-dependent responses (Fig. 4a). Further, these tyrphostins differ greatly in potency and efficacy (Table 1). Tyrphostin-51, tyrphostin-23 and tyrphostin25 acted as full agonists, while others were partial agonists. Searching the DrugBank database (http://www.drugbank.ca/) identified entacapone that is a tyrphostin analogy drug (Fig. 3). Entacapone is a selective, reversible catechol-O-methyl transferase

CN

CN

CN

CN

Since tyrphostin-51 is the most potent ligand among tyrphostins in DMR assays, we examined its ability to cause receptor internalization. Results showed that stimulation of HT29 with 10 lM tyrphostin-51 led to significant internalization of GPR35 (Fig. 5a). Since ERK phosphorylation is a hallmark of the activation and signaling of many GPCRs including GPR35 [16], we examined the ability of both tyrphostin-51 and entacapone to result in ERK phosphorylation. Results showed that stimulation of HT-29 cells with both agonists led to robust phosphorylation of ERK (Fig. 5b). As a control, the known GPR35 agonist zaprinast also led to robust ERK phosphorylation, while tyrphostin-1 was inactive as expected (Fig. 5b). Finally, we examined the ability of tyrphostins to cause arrestin translocation using the Tango™ GPR35 arrestin assays. Results showed that zaprinast led to a dose dependent response in Tango™ GPR35-bla U2OS cells with an EC50 of 6.2 ± 0.9 lM (n = 4) (Fig. 6a; Table 1). Similarly, tyrphostin-51 also led to a comparable response, leading to an EC50 of 7.7 ± 0.9 lM (n = 4) (Fig. 6b), suggesting that similar to zaprinast tyrphostin-51 acted as a full agonist for GPR35

HO

CN

CN CN

O

NH2

NH2

CN

CN

HO

Tyrphostin 9

Tyrphostin 1

AG-370

AG-112

CN

HO

HO

CN

CN

HO CN

HO

O

NH2

CN

CN HO

Tyrphostin 25

C2H5 N

O2N CN

CN

C2H5

HO

HO

OH

Tyrphostin 23

CN

N H

OH

OH

Tyrphostin 51

Entacapone

o

O CONH2 HO

CONH2

CN

HO

N H

CN

N H CN

CN HO

HO

HO

Tyrphostin 47

OH

Tyrphostin 46

HO

HO

AG-490

AG-494

Br

CN

HO

CN

CN

CN

CN

CN

CONH2

N S

CN

S

HO

O2N

HO

OH

NO2 AG-1024

HO

AG-1288

AG-126

O AG-825

Fig. 3. Structures of a series of tyrphostins tested. All ligands, but tyrphostin-1 and AG-490, exhibit agonism activity on GPR35 in HT29 cells.

1961

H. Deng et al. / FEBS Letters 585 (2011) 1957–1962

2000nM 1000nM 500nM 250nM 125nM

200 100

63nM 31nM 16nM

0

c

300 32µM 16µM

250 200

8µM

150 100

4µM

50

2µM 1µM 0.5µM

0

-100

Response (pm)

b

300

Response (pm)

Response (pm)

a

10

20

30

40

50

200 100 0 Entacapone Tyrphostin 51

-100

-50

0

300

0

10

20

Time (min)

30

40

-9

50

-7

-8

Time (min)

-6

-5

-4

Compound, log M

Fig. 4. Characteristics of the DMR signals induced by tyrphostin-51 and entacapone. (a) The dose DMR responses of tyrphostin-51; (b) the dose DMR responses of entacapone; and (c) the DMR amplitudes (8 min post-stimulation) of both tyrphostin-51 and entacapone as a function of ligand doses.

Zaprinast

Tyrphostin-1

Entacapone

Tyrphostin 51

b

DMSO

a

pERK1/2 ERK2

10µm

Actin

Fig. 5. Receptor internalization induced by 10 lM tyrphostin-51 (a) and ERK phosphorylation induced by 10 lM tyrphostin-51, 32 lM entacapone, 32 lM tyrphostin-1 or 1 lM zaprinast (b). The confocal image was obtained after the cells were stimulated with tyrphostin-51 for 1 h, permeabilized and stained with anti-GPR35, followed staining with the fluorescent secondary antibody.

15

Zaprinast Tryphostin-25

12

Tryphostin-47 AG-1288 Tryphostin-1

9

b

15

Zaprinast Tyrphostin-51 Tyrphostin-46 Tyrphostin-23 Entacapone

12

Response ratio

Response (pm)

a

6

9 6 3

3

0

0 -9

-8

-7

-6

-5

-4

-3

-9

-8

-7

-6

-5

-4

-3

Compound, log M

Compound, log M

Fig. 6. Arrestin translocation induced beta-lactamase expression upon the activation of GPR35 by tyrphostins in Tango™ GPR35-bla U2OS cells. The coumarin to fluorescein ratio was plotted as a function of ligand doses. Zaprinast was included as a positive control, while tyrphostin-1 as a negative control.

to cause arrestin translocation. Interestingly, several other tyrphostins including tyrphostin-23, tyrphostin-25, tyrphostin-46, tyrphostin-47 and AG-1288 acted as partial agonists (Fig. 6), while other tyrphostins tested were inactive. It is worthy noting that the Tango arrestin assay generally led to a right shift in potency. Nonetheless, these results suggest that tyrphostins including entacapone are GPR35 agonists.

4. Discussion GPR35 is a poorly-characterized orphan GPCR with a few known agonists. To identify GPR35 ligands, we first showed that GPR35 is present and located primarily at the cell surface of native HT29 cells (Fig. 1d). DMR assays further showed that endogenous GPR35 in HT29 is functional, whose activation by three known GPR35 agonists

1962

H. Deng et al. / FEBS Letters 585 (2011) 1957–1962

leads to robust DMR signals (Fig. 1a and b). Screening three kinase inhibitor libraries using DMR agonism assay led to identification of 14 tyrphostin analogs that acted as GPR35 agonists. Among all tyrphostins examined, tyrphostin-51 was found to be the most potent ligand with an EC50 of 190 nM (Fig. 4a), which is comparable to the potency of zaprinast. Tyrpostin-51 resulted in significant internalization of GPR35, the degree of which is comparable to that induced by pamoic acid (Fig. 5a). Further, tyrophstin-51 also led to robust ERK phosphorylation (Fig. 5b) and b-arrestin translocation (Fig. 6b). Tyrphostins were originally designed and developed for inhibiting tyrosine kinases [19]. Like all kinase inhibitors tyrphostins often interact with multiple targets. Thus, the identification of tyrphostins as potent GPR35 agonists adds new target class to these kinase inhibitors. One of the most notable findings of this study is that entacapone was identified to be a GPR35 agonist with a moderate potency. Chemical similarity searching of the Drug Bank database allowed us to identify entacapone as a tyrphostin analog drug. Follow-up DMR and receptor signaling assays showed that (1) entacapone resulted in a DMR similar to other GPR35 agonists; (2) entacapone caused cells desensitized upon the repeated stimulation with zaprinast; and (3) entacapone behaved similarly to tyrphostin-51 in receptor signaling assays. Entacapone was apparently inactive in the Tango™ arrestin gene reporter assays within the concentration range examined. However, due to the right shift in potency in the arrestin assays, we cannot rule out the possibility of entacapone to be a weak agonist in causing arrestin translocation. Entacapone is a nitrocatechol drug that is used in the treatment of Parkinson’s disease in conjunction with dopaminergic agents. The primary mechanism of action is believed to be that entacapone acts as a CMOT inhibitor, thus preventing COMT from metabolizing L-DOPA into 3methoxy-4-hydroxy-L-phenylalanine in the periphery, which does not easily cross the blood–brain barrier. The moderate potency of entacapone observed here raises an intriguing point whether its GPR35 agonism activity is relevant to its clinical features. In conclusion, the ligands identified here provide new pharmacological tools to characterize the biology and pharmacology of GPR35. The present study also highlights the power of label-free DMR assays to discover new ligands for poorly characterized receptors, including GPR35. References [1] O’Dowd, B.F., Nguyen, T., Marchese, A., Cheng, R., Lynch, K.R., Heng, H.H., Kolakowski Jr., L.F. and George, S.R. (1998) Discovery of three novel G-proteincoupled receptor genes. Genomics 47, 310–313.

[2] Okumura, S., Baba, H., Kumada, T., Nanmoku, K., Nakajima, H., Nakane, Y., Hioki, K. and Ikenaka, K. (2004) Cloning of a G-protein-coupled receptor that shows an activity to transform NIH3T3 cells and is expressed in gastric cancer cells. Cancer Sci. 95, 131–135. [3] Imielinski, M. et al. (2009) Common variants at five new loci associated with early-onset inflammatory bowel disease. Nat. Genet. 41, 1335–1340. [4] Yang, Y., Lu, J.Y.L., Wu, X., Summer, S., Whoriskey, J., Saris, C. and Reagan, J.D. (2010) G-protein-coupled receptor 35 is a target of the asthma drugs cromolyn disodium and nedocromil sodium. Pharmacology 86, 1–5. [5] Sparfel, L., Pinel-Marie, M.-L., Boize, M., Koscielny, S., Desmots, S., Pery, A. and Fardel, O. (2010) Transcriptional signature of human macrophages exposed to the environmental contaminant benzo(a)pyrene. Toxicol. Sci. 114, 247–259. [6] Min, K.D., Asakura, M., Liao, Y., Nakamaru, K., Okazaki, H., Takahashi, T., Fujimoto, K., Ito, S., Takahashi, A., Asanuma, H., Yamazaki, S., Minamino, T., Sanada, S., Seguchi, O., Nakano, A., Ando, Y., Otsuka, T., Furukawa, H., Isomura, T., Takashima, S., Mochizuki, N. and Kitakaze, M. (2010) Identification of genes related to heart failure using global gene expression profiling of human failing myocardium. Biochem. Biophys. Res. Commun. 393, 55–60. [7] Wang, J., Simonavicius, N., Wu, X., Swaminath, G., Reagan, J., Tian, H. and Ling, L. (2006) Kynurenic acid as a ligand for orphan G protein-coupled receptor GPR35. J. Biol. Chem. 281, 22021–22028. [8] Oka, S., Ota, R., Shima, M., Yamashita, A. and Sugiura, T. (2010) GPR35 is a novel lysophosphatidic acid receptor. Biochem. Biophys. Res. Commun. 395, 232–237. [9] Milligan, G. (2011) Orthologue selectivity and ligand bias: translating the pharmacology of GPR35. Trends Pharmacol. Sci. 32, 317–325. [10] Fang, Y. (2010) Label-free receptor assays. Drug Discov. Today Technol. 7, e5– e11. [11] Fang, Y., Ferrie, A.M., Fontaine, N.H., Mauro, J. and Balakrishnan, J. (2006) Resonant waveguide grating biosensor for living cell sensing. Biophys. J. 2006 (91), 1925–1940. [12] Kenakin, T. (2009) Cellular assays as portals to seven-transmembrane receptor-based drug discovery. Nat. Rev. Drug Discov. 8, 617–626. [13] Schröder, R., Janssen, N., Schmidt, J., Kebig, A., Merten, N., Hennen, S., Müller, A., Blättermann, S., Mohr-Andrä, M., Zahn, S., Wenzel, J., Smith, N.J., Gomeza, J., Drewke, C., Milligan, G., Mohr, K. and Kostenis, E. (2010) Deconvolution of complex G protein-coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nat. Biotechnol. 28, 943–949. [14] Fang, Y. (2010) Probing cancer signaling with resonant waveguide grating biosensors. Exp. Opin. Drug Discov. 5, 1237–1248. [15] Taniguchia, Y., Tonai-Kachia, H. and Shinjo, K. (2006) Zaprinast, a well-known cyclic guanosine monophosphate-specific phosphodiesterase inhibitor, is an agonist for GPR35. FEBS Lett. 580, 5003–5008. [16] Zhao, P., Sharir, H., Kapur, A., Cowan, A., Geller, E.B., Adler, M.W., Seltzman, H.H., Reggio, P.H., Heynen-Genel, S., Sauer, M., Chung, T.D.Y., Bai, Y., Chen, W., Caron, M.G., Barak, L.S. and Abood, M.E. (2010) Targeting of the orphan receptor GPR35 by pamoic acid: a potent activator of ERK and b-arrestin2, with antinociceptive activity. Mol. Pharmacol. 78, 560–568. [17] Jenkins, L., Brea, J., Smith, N.J., Hudson, B.D., Reilly, G., Bryant, N.J., Castro, M., Loza, M.I. and Milligan, G. (2010) Identification of novel species-selective agonists of the G-protein-coupled receptor GPR35 that promote recruitment of b-arrestin-2 and activate Ga13. Biochem. J. 432, 451–459. [18] Wishart, D.S., Knox, C., Guo, A.C., Cheng, D., Shrivastava, S., Tzur, D., Gautam, B. and Hassanali, M. (2007) DrugBank: a knowledgebase for drugs, drug actions and drug targets. Nucleic Acids Res. 36, D901–D906. [19] Levitzki, A. and Mishani, E. (2006) Tyrphostins and other tyrosine kinase inhibitors. Annu. Rev. Biochem. 75, 93–109.